Designing artificial extracellular matrices that effectively signal and direct cellular responses is essential for the creation of new regenerative medicine therapies. However, the preparation of any synthetic material with structure on the nanoscale that mimics natural tissue is a challenging problem. One approach has been to prepare molecules that spontaneously assemble into fibers similar in morphology to the proteins and proteoglycans that compose the natural extracellular matrix. In contrast to most synthetic biopolymers, the use of small, self-assembling molecules facilitates control of chemical and structural properties of these macromolecular assemblies. Injectable, self-assembling biomaterials capable of forming scaffolds in situ around cells are promising therapeutic candidates because of their minimally invasive delivery. The signaling efficiency of self-assembling bioactive structures depends not only on molecular structure but also on nanoscale morphology. To that end, peptide amphiphiles have been shown to self-assemble under suitable conditions to form nanoscale aggregates having particular utility as biocompatible scaffolds, more particularly in the area of tissue engineering and regenerative medicine.
Peptide amphiphiles (PAs) constitute a class of molecules that spontaneously self-assemble into a variety of nanostructures, including spherical micelles, cylindrical fibers and ribbons, and have shown promising therapeutic activity. PA molecules that form cylindrical fiber-like nanostructures (termed in the art “nanofibers”) have been found to be particularly useful in mimicking the biomechanical properties of the extracellular matrix. These PA molecules are composed typically of four main segments: (1) a hydrophobic moiety, commonly an acyl group derived from a fatty acid, that promotes molecular aggregation through hydrophobic collapse; (2) a β-sheet-forming peptide that promotes fiber assembly; (3) a peptide segment that contains ionizable side-chain residues; and (4) a peptide signaling moiety designed to interact with cellular receptors. These molecules self-assemble into high-aspect ratio nanostructures, forming gels in water at low concentrations when the charges on the ionic side chains are appropriately screened. Individual cylindrical nanofibers display bioactive sequences on their surface in high density. However, aggregation of nanofibers into bundles reduces the surface area for bioactive epitope presentation. Moreover, some PA molecules have proven difficult to synthesize and/or purify on a large scale. This can be due to the molecules' zwitterionic nature (i.e., carrying both positive and negative charges), and their propensity to aggregate in solution due to the relative large proportion of non-polar amino acid residues.
Peptide amphiphiles bearing the laminin-derived peptide sequence IKVAV (SEQ ID NO. 1) have been reported in the prior art to selectively differentiate neural stem cells into neurons, to promote neurite outgrowth, and to suppress differentiation into astrocytes in vitro. Furthermore, IKVAV-bearing peptide amphiphiles have been reported to promote axonal regeneration, improve motor function, reduce apoptosis and reduce astrogliosis in vivo in spinal cord lesions following spinal cord injury (SCI) in rodents. In addition, therapeutics incorporating the IKVAV (SEQ ID NO. 1) peptide may be used to treat other neurodegenerative conditions such as Parkinson's and Alzheimer's disease, by promoting regeneration of neurons from endogenous or exogenous neural stem cells. These compounds may also be used to minimize tissue damage or promote repair or regenerate of brain tissue following cerebral trauma or stroke.
The IKVAV (SEQ ID NO. 1) epitope has been shown to bind to at least two receptors, a 110 kDa laminin binding protein (LBP110/APP) and nucleolin, although the exact molecular arrangement of this binding and the signal transduction pathways are not known from the prior art. The IKVAV (SEQ ID NO. 1) peptide contains four hydrophobic amino acids with a strong propensity to form beta-sheet secondary and tertiary structures.
Peptides containing this epitope are reported to form amyloid-like fibrils. If the IKVAV (SEQ ID NO. 1) peptide epitope is hydrogen bonded in a rigid beta-sheet conformation with neighboring epitopes, this will likely reduce its ability to bind to target cellular receptors. While the IKVAV (SEQ ID NO. 1) peptide itself is known to promote neurite sprouting, in previous studies on IKVAV-bearing polymer scaffolds, enhanced neurite outgrowth and neuronal differentiation were not observed, possibly due to reduced epitope presentation resulting from unfavorable aggregation.
Thus, there exists a technological need, unfulfilled by the prior art, for an effective supramolecular strategy to control epitope presentation of hydrophobic bioactive peptides such as IKVAV (SEQ ID NO. 1) to enhance signal transduction. Biologically active IKVAV-bearing PA molecules that may be purified using standard high performance liquid chromatography (HPLC) techniques are also needed. There is furthermore a need to develop IKVAV-bearing PAs that are soluble in salt solutions similar to extracellular fluid, in order to facilitate injection of PA solutions into living tissue.